The field of this disclosure relates to aging-associated oxidative damage, and specifically, to an aging-specific particle associated Lipoprotein B NADH oxidase (apoBNOX) attached to circulating lipoprotein particles, its use as a target to identify agents for the prevention or treatment of disorders caused by oxidative damage to circulating lipoproteins and the identification of such agents for use as dietary supplements and/or pharmaceutical preparations with examples derived from olive oil and white wine, including tyrosol (4-hydroxyphenethyl alcohol) and its hydroxylated derivative, hydroxytyrosol.
apoBNOX is a member of the ECTO-NOX or ENOX family of proteins. These proteins exhibit one or more cyanide-resistant external plasma membrane or soluble hydroquinone oxidases capable of catalyzing protein disulfide interchange and that oxidize NAD(P)H as an alternate substrate (NADH oxidase=NOX) (Morré, 1998, in Plasma Membrane Redox Systems and Their Role in Biological Stress and Disease, H. Asard, A. Bérczi and R. J. Caubergs, eds., pp. 121-156, Kluwer Academic Publishers, Dordrecht, Netherlands; Morré and Morré, 2003, Free Radical Res. 37: 795-808). Based on activity characteristics, at least three but distinct ECTO-NOX or ENOX (for external cell surface NOX) proteins have been described. These proteins are characterized by the property of having two distinct biochemical activities, hydroquinone (NAD(P)H) oxidation and protein disulfide-thiol interchange, that alternate. One (CNOX or ENOX1) is constitutive with an activity that is widely distributed among animals, plants and yeasts (Jiang et al., 2008, Biochemistry 48: 14018-14038). A second ENOX activity is tumor or cancer-associated, designated tNOX or ENOX2 (Hostetler et al., 2009, Clin. Proteomics 5: 46-51). ENOX 2 proteins are inhibited by a series of quinone site inhibitors all with anticancer activity. A third protein with ENOX-like activity (designated arNOX) is age-related (Morré et al., 2003, Mol. Cell. Biochem. 254: 101-109) and predominant only on cell surfaces and body fluids of aged individuals and on plasma membrane of late passage cultured cells or senescent plant parts. Shed forms of both activities are found in body fluids and in culture media conditioned by the growth of mammalian or plant cells. arNOX differs from ENOX1 and ENOX2 by generating superoxide based on superoxide susceptible oxidation of ferricytochrome c, a standard method for measurement of superoxide generation (Butler et al., 1982, J. Biol. Chem. 257: 10747-10750).
apoBNOX differs from ENOX1 and ENOX2 as well as the canonical arNOX by being tightly bound to circulating low density lipoprotein (LDL) particles and carrying out ferricytochrome c reduction which is resistant to inhibition by superoxide dismutase. It also exhibits an inhibitor response pattern different from other ENOX proteins and utilizes lipoprotein particle-bound protein B as its natural substrate. The activity of arNOX, which differs in the above important respects from apoBNOX in aging cells and in sera, has been described previously (Morré and Morré, 2006, Rejuvenation Res. 9: 231-236).
Age and oxidative stress are major risk factors for heart disease (Schmuck et al., 1995, Clin. Chem. 41: 1628-1632). A large body of evidence supports the notion that reactive oxygen species provide a causal link in the appearance of oxidized circulating lipoproteins such as oxidized LDLs and their subsequent clearance by macrophages and delivery to the arterial wall. It now appears likely that oxidized LDL is a major contributor to progressive atherogenesis by enhancing endothelial injury, by inducing foam cell (lipoprotein engorged macrophages) generation and associated smooth muscle proliferation (Halvoet, 1999, Ther. Apher. 3: 287-293). Macrophages clear the circulation of oxidized lipoprotein particles by internalizing them and in so doing are transformed into foam cells. The foam cells deliver their cargo of oxidized fats and cholesterol where they are deposited beneath the arterial wall. Such progressive delivery of oxidatively-damaged lipoprotein particles eventually leads to atherosclerotic plaques and advanced heart disease.
However, the basis for LDL oxidation has been little studied. Levels of common antioxidants including α-tocopherol, β-carotene and ascorbate decline with age but there is no apparent correlation between ingestion of these common antioxidants and amelioration of the aging process or decreased mortality (Bjelakovic et al., 2007, JAMA 294: 842-857). The implication is that the oxidative damage leading to aging and increased atherogenic risk is the result of a much more specific causation. Why does LDL oxidation increase in the elderly and why is it greater in some individuals than in others? Our findings suggest that LDL oxidation in the elderly and in individuals at high risk for heart disease correlates with levels of circulating apoBNOX.
The amount of apoBNOX associated with circulating lipoprotein particles increases starting at about age 30 and reaches maximum at about age 75 in males and age 55-65 in females. Of those who die of a heart attack, 85% are 65 or older (American Heart Association, 2008, Circulation 17: e25-3146). Women surviving beyond age 65 usually have diminished apoBNOX levels compared to men and a lower risk of cardiovascular disease compared to men (Kannel and Lavine, 2003, Prog. Cardiovasc. Nursing 18: 135-140) further suggesting some causal relationship between apoBNOX levels and atherogenic risk.
Consequently, there is a need to find agents that inhibit apoBNOX for the purposes of reducing or treating the resultant physiological conditions, such as oxidation of apoprotein B molecules in low density lipoprotein (LDLs) and attendant arterial changes. The arNOX activity of aging cells has been shown to be inhibited by naturally occurring agents such as coenzyme Q (ubiquinone) including CoQ10, CoQ9 and CoQ8 (Morré et al., 2008, BioFactors 32: 231-235); Morré et al., 2003, Mol. Cell. Biochem. 254: 101-109; U.S. Pat. No. 6,878,514). These agents, however, are relatively ineffective in the inhibition of apoBNOX.
Even if it were an effective inhibitor, the use of coenzyme Q would not completely be satisfactory as an apoBNOX inhibitor for several reasons: it is costly, it oxidizes easily losing its efficacy, and preparations containing coenzyme Q must be specially packaged to prevent loss of function. Thus, there are no economical and chemically stable agents or methods currently known to inhibit apoBNOX activity. Accordingly, it would be an improvement in the art to identify economical and chemically stable agents and techniques with the agents that inhibit apoBNOX but that are also non-toxic and advantageously are naturally occurring.